The present invention relates to a vaccine formula allowing the vaccination of dogs against a large number of infectious pathologies, in particular respiratory and digestive pathologies. It also relates to a corresponding method of vaccination.
Infectious dog pathology is extremely varied and often difficult to control depending on the circumstances encountered in the field.
A number of vaccines already exist, in particular against Carrxc3xa9""s disease (CDV virus), parvovirosis (CPV virus), coronavirosis (CCV virus) kennel cough or respiratory complex (PI2 virus) and rabies (rhabdovirus). These vaccines are, more generally, live vaccines consisting of attenuated strains. This is especially the case for Carrxc3xa9""s disease vaccines, vaccines against canine adenoviroses, vaccines against parvovirosis and vaccines against the canine coronavirus.
In some cases, inactivated vaccines have also been proposed, as for rabies and coronavirosis.
These various vaccines are sold either separately, that is to say in the form of monovalent vaccines, or in the form of associated, that is to say polyvalent, vaccines.
The polyvalent associations developed up until now have always posed problems of compatibility between the valencies and of stability. It is indeed necessary to ensure at the same time the compatibility between the different valencies of the vaccine, whether from the point of view of the different antigens used or from the point of view of the formulations themselves, especially in the case where both inactivated vaccines and live vaccines are combined. It also poses the problem of preservation of such combined vaccines and also of their safety especially in the presence of adjuvant. These vaccines are in general quite expensive.
The degree of protection and the duration of this protection can, in addition, be highly variable and are also sensitive to the circumstances in the field. This is particularly true of the vaccination of puppies, in which the antibodies of maternal origin prevent immunization by the inactivated vaccines and even by live vaccines.
It may therefore be desirable to perfect the vaccination of Canidae, and especially dogs, while keeping in mind the economic constraints acting against the use of vaccines which are expensive or complicated to use.
Vaccination trials against Carrxc3xa9""s disease using purified preparations of F fusion antigens and of H haemaglutinin equivalents in complete Freund""s adjuvant have suggested that the F antigen might constitute an immunogen of interest for protection against the CDV virus (E. Norrby et al., J. of Virol. May 1986: 536-541) for a subunit vaccine.
Another article (P. de Vries et al., J. gen. Virol. 1988, 69: 2071-2083) suggests, on the other hand, that the CDV F and HA proteins might be advantageous in a vaccination according to the technique of immunostimulatory complexes (ISCOMS).
Mice immunized with a recombinant vaccine expressing the gene for the CDV F protein were protected against challenge with this virus.
These are, however, laboratory results, which are difficult to interprete especially under field conditions.
As regards parvoviroses, trials of subunit vaccines containing the major capsid protein VP2 from the CPV virus obtained by genetic recombination in the baculovirus made it possible to show protection of dogs thus immunized against challenge with the CPV virus.
As regards the canine herpesvirus CHV, studies have been carried out on the use of glycoproteins as components of subunit vaccines. These studies have shown the induction of cross-responses with other herpesviruses such as FHV but do not draw any conclusion on the possibilities of making a protective vaccine.
For the Lyme disease, associated OspA and OspB induce protection in mice and dogs and OspA alone in mice, hamsters and dogs.
Patent applications WO-A-90 11092, WO-A-93 19183, WO-A-94 21797 and WO-A-95 20660 have made use of the recently developed technique of polynucleotide vaccines. It is known that these vaccines use a plasmid capable of expressing, in the host cells, the antigen inserted into the plasmid. All routes of administration have been proposed (intraperitoneal, intravenous, intramuscular, transcutaneous, intradermal, mucosal and the like). Various means of vaccination can also be used, such as DNA deposited at the surface of gold particles and projected so as to penetrate into the animal""s skin (Tang et al., Nature 356, 152-154, 1992) and liquid jet injectors which make it possible to transfect the skin, muscle, fatty tissues as well as the mammary tissues (Furth et al., Analytical Biochemistry, 205, 365-368, 1992). (See also U.S. Pat. Nos. 5,846,946, 5,620,896, 5,643,578, 5,580,589, 5,589,466, 5,693,622, and 5,703,055; Science, 259:1745-49, 1993; Robinson et al., seminars in IMMUNOLOGY, 9:271-83, 1997; Luke et al., J. Infect. Dis. 175(1):91-97, 1997; Norman et al., Vaccine, 15(8):801-803, 1997; Bourne et al., The Journal of Infectious Disease, 173:800-7, 1996; and, note that generally a plasmid for a vaccine or immunological composition can comprise DNA encoding an antigen operatively linked to regulatory sequences which control expression or expression and secretion of the antigen from a host cell, e.g., a mammalian cell; for instance, from upstream to downstream, DNA for a promoter, DNA for a eukaryotic leader peptide for secretion, DNA for the antigen, and DNA encoding a terminator.)
The polynucleotide vaccines may use both naked DNAs and DNAs formulated, for example, inside liposomes or cationic lipids.
The prior art, on the other hand, gives no result of protection in dogs by the polynucleotide method of vaccination against these diseases. Much less is yet known about the canine coronavirus CCV and about the agents responsible for the respiratory complex.
As regards rabies, protection of mice against virulent challenge has been demonstrated after treatment with a polynucleotide vaccine expressing the gene for the G protein under the control of the SV40 virus early promoter (Xiang et al., Virology 199, 1994: 132-140), a similar result being achieved by using the CMV IE promoter.
The invention proposes to provide a multivalent vaccine formula which makes it possible to ensure vaccination of dogs against a number of pathogenic agents.
Another objective of the invention is to provide such a vaccine formula combining different valencies while exhibiting all the required criteria of mutual compatibility and stability of the valencies.
Another objective of the invention is to provide such a vaccine formula which makes it possible to combine different valencies in the same vehicle.
Another objective of the invention is to provide such a vaccine formula which is easy and inexpensive to use.
Yet another objective of the invention is to provide a method of vaccination which makes it possible to considerably increase the efficacy of the vaccine according to the invention or to substantially reduce the quantity of vaccine necessary, and having good safety.
The subject of the present invention is therefore a vaccine formula against Canidae pathogens, comprising at least two vaccine valencies each comprising a plasmid integrating, so as to express it in vivo in the Canidae cells, a gene with one canine pathogen valency, namely a Carrxc3xa9""s disease virus CDV valency and a canine parvovirus CPV valency, the plasmids comprising, for each valency, one or more of the genes selected from the group consisting of HA and F for the Carrxc3xa9""s disease virus and the VP2 gene for the canine parvovirus.
Preferably, for the Carrxc3xa9""s disease valency, the plasmid(s) comprise the HA and F genes, either inserted into the same plasmid, or inserted into different plasmids.
The multivalent vaccine according to the invention may also comprise a canine coronavirus CCV valency, with one or several plasmids comprising one or more of the genes selected from the group of the S and M genes and preferably the S gene or the S and M genes. Here also, the genes may be inserted into different plasmids or grouped together in the same plasmid in a context allowing their expression. The abovementioned bi- or trivalent vaccine according to the invention may also comprise, in addition, a valency effective for the prevention of the respiratory complex, namely a PI2 valency comprising one or several plasmids which comprise at least one of the HA and F genes. Preferably, the use of both the two HA and F genes is envisaged.
Other advantageous valencies in the case of the present invention may therefore be associated with the vaccines according to the invention, namely one or more of the valencies selected from the group formed by the herpesvirosis CHV, Lyme disease and rabies, the plasmids comprising, for each valency, one or more of the genes selected from the group composed of the gB and gD genes for the CHV virus, the OspA, OspB and p100 genes for B. burgdorferi (Lyme disease), and the G gene for rabies.
Preferably, for herpesvirosis, the two gB and gD genes are associated either in two separate plasmids, or in a single plasmid. For Lyme disease, the OspA gene is preferred.
Preferably, the vaccine according to the invention comprising the Carrxc3xa9""s disease and parvovirosis valencies will comprise, as other valency, the coronavirosis valency or, less preferably, the respiratory complex valency, or these two valencies, it being understood that any combination comprising, one, several or all the coronavirosis, respiratory complex, herpesvirosis, Lyme disease and rabies valencies can be associated with the two Carrxc3xa9""s disease and parvovirosis valencies.
Valency in the present invention is understood to mean at least one antigen providing protection against the virus for the pathogen considered, it being possible for the valency to contain, as subvalency, one or more modified or natural genes from one or more strains of the pathogen considered.
Pathogenic agent gene is understood to mean not only the complete gene but also the various nucleotide sequences, including fragments which retain the capacity to induce a protective response. The notion of the gene covers the nucleotide sequences equivalent to those described precisely in the examples, that is to say the sequences which are different but which encode the same protein. It also covers the nucleotide sequences of other strains of the pathogen considered, which provide cross-protection or a protection specific for a strain or for a strain group. It also covers the nucleotide sequences which have been modified in order to facilitate the in vivo expression by the host animal but encoding the same protein.
The different valencies are contained in the vaccinal formulation according to the invention in a therapeutically effective quantity.
Preferably, the vaccine formula according to the invention can be provided in a suitable vehicle for administration, preferably by the intramuscular route, in a dose volume of between 0.1 and 5 ml, preferably between 0.5 and 2 ml.
The dose will be generally between 10 ng and 1 mg, preferably 100 ng and 500 xcexcg, and preferably between 1 xcexcg and 250 xcexcg per plasmid type.
Use will preferably be made of naked plasmids simply placed in the vaccination vehicle which will be in general physiological saline (0.9% NaCl), ultrapure water, TE buffer and the like. All the polynucleotide vaccine forms described in the prior art can of course be used.
Each plasmid comprises a promoter capable of ensuring the expression of the gene inserted, under its control, into the host cells. This will be in general a strong eukaryotic promoter and in particular a cytomegalovirus early CMV-IE promoter of human or murine origin, or optionally of another origin such as rats, pigs and guinea pigs.
More generally, the promoter may be either of viral origin or of cellular origin. As viral promoter other than CMV-IE, there may be mentioned the SV40 virus early or late promoter or the Rous sarcoma virus LTR promoter. It may also be a promoter from the virus from which the gene is derived, for example the gene""s own promoter.
As cellular promoter, there may be mentioned the promoter of a cytoskeleton gene, such as for example the desmin promoter (Bolmont et al., Journal of Submicroscopic Cytology and Pathology, 1990, 22, 117-122; and Zhenlin et al., Gene, 1989, 78, 243-254), or alternatively the actin promoter.
When several genes are present in the same plasmid, these may be presented in the same transcription unit or in two different units.
The combination of the different vaccine valencies according to the invention may be preferably achieved by mixing the polynucleotide plasmids expressing the antigen(s) of each valency, but it is also possible to envisage causing antigens of several valencies to be expressed by the same plasmid.
The subject of the present invention is also a method for vaccinating dogs, comprising the administration of an effective dose of a vaccine formula as described above. This vaccination method comprises the administration of one or more doses of the vaccine formula, it being possible for these doses to be administered in succession over a short period of time and/or in succession at widely spaced intervals.
The vaccine formulae according to the invention can be administered in the context of this method of vaccination, by the different routes of administration proposed in the prior art in the case of polynucleotide vaccination and by means of known techniques of administration, the preferred route being the intramuscular route.
The efficiency of presentation of the antigens to the immune system varies according to the tissues. In particular, the mucous membranes of the respiratory tree serve as barrier to the entry of pathogens and are associated with lymphoid tissues which support local immunity. The administration of a vaccine by contact with the mucous membranes, in particular the buccal mucous membrane, the pharyngeal mucous membrane and the mucous membrane of the bronchial region, is certainly of interest for vaccination against respiratory and digestive pathologies.
Consequently, the mucosal routes of administration form part of a mode of administration for the invention using in particular nebulization or spray or drinking water. It will be possible to apply the vaccine formulae and the vaccination methods according to the invention in this content.
The subject of the invention is also monovalent vaccine formulae comprising one or more plasmids encoding one or more genes from one of the viruses above, the genes being those described above. Besides their monovalent character, these formulae may possess the characteristics stated above as regards the choice of the genes, their combinations, the composition of the plasmids, the dose volumes, the doses and the like.
The monovalent vaccine formulae may be used (i) for the preparation of a polyvalent vaccine formula as described above, (ii) individually against the actual pathology, (iii) combined with a vaccine of another type (live or inactivated whole, recombinant, subunit) against another pathology, or (iv) as booster for a vaccine as described below.
The subject of the present invention is in fact also the use of one or more plasmids according to the invention for the manufacture of a canine vaccine intended to vaccinate animals first vaccinated by means of a first conventional vaccine (monovalent or multivalent) of the type in the prior art, in particular selected from the group consisting of a live whole vaccine, an inactivated whole vaccine, a subunit vaccine, a recombinant vaccine, this first vaccine having (that is to say containing or capable of expressing) the antigen(s) encoded by the plasmid(s) or antigen(s) providing cross-protection.
Remarkably, the polynucleotide vaccine has a potent booster effect which results in an amplification of the immune response and the acquisition of a long-lasting immunity.
In general, the first-vaccination vaccines can be selected from commercial vaccines available from various veterinary vaccine producers.
The subject of the invention is also the method of vaccination consisting in making a first vaccination as described above and a booster with a vaccine formula according to the invention.
In a preferred embodiment of the process according to the invention, there is administered in a first instance, to the animal, an effective dose of the vaccine of the conventional, especially inactivated, live, attenuated or recombinant type, or alternatively a subunit vaccine, so as to provide a first vaccination, and, after a period preferably of 2 to 6 weeks, the polyvalent or monovalent vaccine according to the invention is administered.
The subject of the invention is also a vaccination kit grouping together a first-vaccination vaccine as described above and a vaccine formula according to the invention for the booster. It also relates to a vaccine formula according to the invention accompanied by a leaflet indicating the use of this formula as a booster for a first vaccination as described above.
The invention also relates to the method of preparing the vaccine formulae, namely the preparation of the valencies and mixtures thereof, as evident from this description.
The invention will now be described in greater detail with the aid of the embodiments of the invention taken with reference to the accompanying drawings.